Dedicated to the Cargo Cults of Biology Science, Biotechnology and the Pharmaceutical Industry.
"So we really ought to look into theories that don't work, and science that isn't science"
Richard Feynman,
Cargo Cult Science,
From a Caltech commencement address given in 1974

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Friday, June 30, 2006

There is a technique that is used in biotechnology known as phage display. In this technology a virus that attacks bacteria is used to display a short peptide. Without boring the reader with the details, this is a technique known as evolution in an instant. What you do is subject a library of peptide displaying phage to a protein target. You wash away non-binding phage, amplify binding phage and repeat. What you hope to end up with are peptides that bind to a target. You are repeating because it takes a while to get rid of non-binding phage. They must be slowly selected against in an evolution like process.

During the selection process many other things can happen. Contamination of the library of phage can happen. Most interesting however is the selection of phage that are in the original library. These phage will predominate after a while because they grow faster than the other phage. At least that is the theory. A company called New England Biolabs sells the phage libraries. Each lot appears to have a set of contaminants that will come out after a period of selection. They do not seem to appear because they bind to a protein target. They lack a gene that the rest of the library has. This gene causes the phage plaques (formed on bacterial lawns) to turn blue. The contaminating phage form white plaques. Perhaps this is part of the selection of the phage since the normal state of the DNA lacks the blue plaque gene.

One such phage that has been seen displays the peptide GETRAPL. That is code for 7 amino acids, glycine-glutamic acid-threonine-arginine-alanine-proline-leucine. A co-worker of mine found this sequence. I recognized it and ran it in our database. Sure enough, it showed up several times. There was not particular protein linked to it however. It was linked to later stages of selection however. We noted that the plaques were white in all cases. We typed it into Google and found an interesting group of papers.

Researchers at a different laboratories had found this sequence while using phage display against their targets. The first paper "Design and Assay of Inhibitors of HIV-1 Vpr Cell Killing and Growth Arrest Activity Using Microbial Assay Systems". The next, "Development of efficient viral vectors selective for vascular smooth muscle cells". Completely unrelated, yet they used the peptide to validate their work. They published and ...?

In physics you set up a system. In that system many things are happening. Pressure changes, concentration of molecules changes, heat is given off and so on. One of the question you can ask about a system is whether or not work has been done at a certain period of time. For example, you load a box onto a conveyor belt that takes the box and drops it onto another conveyor belt that puts the box back you to load onto the first conveyor belt. You are sweating. Boxes are moving but only in a circle. Work is not really getting done.

The GETRAPL story is like that. Did work get done? Did anything scientific get done? The papers on GETRAPL appear to have ended. No one is talking about the peptide but there was a time when this little contaminant from a phage display library made two labs get out their typewriters and type of the story of how they got the peptide and what they did with it. And it did something! The circle here is starting with the library, getting the same sequence, publishing and moving on to something else. The way to stop the non-working cycle is to publish a paper on the sequence and let people know when they are being misled. The system here is science as it is being practiced by PhD scientists and journal editors. The people who sell the libraries can also stop the non-work cycle. The system is not producing work.

Feynman said that he hoped we learned something during our educations. Something you can't teach. You can easily learn how a phage library is made and used. You can learn some computer software that will help you assess the peptides you end up with. Finding a protein binding peptide is a rare event, but that is not what is taught. To me, that is the interesting thing about the technique. It is just one of many techniques that can be used to study a protein and it's interactions. You can learn all about the protein prior to using phage display. You can get up a give a talk to the Academy of Sciences on the details of your work. But in the end you have to look at your results. You have to use something inside of you that is not taught. Critical thinking perhaps. Can it really be binding to the target? How can I prove that? How can I disprove that? How can I be sure of any result?

One way of looking at GETRAPL is very obvious to laboratory people. You look at the DNA sequences of each phage that displays GETRAPL. Are they identical or are there various sequences using different codons that code for GETRAPL. This would be strong evidence that it is not being selected for because the original phage grows faster than the rest of the library. Another modern day trick that we applied was to Google the sequence and see if anyone could back up your hopes and dreams that it binds to a specific target. Your education can teach you about the tools. It is up to you to use them.

There are many phage like GETRAPL. There is a paper published by one of the leading phage display scientists that discusses their existance. What is selecting these phage is still unknown. Developing an explanation and proving it is science. There are scientists who currently work on software that can take a list of sequences that come out of a phage display selection process and help you understand what you've got. If you sequence 100 phage and they are all the same sequence for example, the computer only sees one sequence. The computer is weeding out repeated sequences because it indicates selection based on non-binding factors such as faster growing phage. The computer software continues to add new features to help you find binding phage. Someday you could simply put files into this software and it will give you a list of possible binding targets. It could search your own database as well those on the internet or in Pubmed. This is still not going to tell you the whole story. You have to develop an assay. They teach you about assays. The assays however, give you another set of data that you have to interpret. This is what Feynman was hoping you would know how to do. Think scientifically. Ignore your hopes and dreams of getting published and moving on to more interesting projects. Don't act on your desire to take that peptide and fuse it into some elaborate molecule you designed and toss that into an elaborate assay and prove that you can cure AIDS! Slow down. Is the data really saying what you think it is. Think critically and proceed logically.

There are many other peptides that have been discovered that are mere contaminants. The claims that their finders have made on their behalf are quite fancy. Big science words were used and put together in a way that is certainly feasible. No work was done however. Just words used to describe a set of letters that represent amino acids. What is really going on can open doors. What is really going on can make the planes land! But they fade away, these research projects. No resolution. No work was done. No planes landed.

Wednesday, June 28, 2006

I watched an old documentary on Ben Franklin last night. As a young man he owned and operated print shops and newspapers and made a lot of money at it. He was editor, writer, and printer. Currently, newspaper editors go to meetings and review union dispute contracts and so on. The point is that people did not have as much information to deal with back then. Ben Franklin could do all of the above jobs because each one was managable. As populations grow and industries grow more details get spread out to the point that no one really knows the whole story.

After Franklin turned 42 he decided to quit running his businesses and focus his time on science. This was during a time when most scientists were men of liesure. They were amatuers who were amusing themselves by observing nature and trying to discover something no one had yet discovered. Franklins field was electricity. Like other amatuer scientists he had to make much of his own equipment. He relied upon his understanding of electricity to create electrodes and electron generators. He ended up getting famous from harnessing this energy and telling about it in a manner far different than the way scientists talk today. He gave us the lightning rod which is still in use today, unchanged from Franklins time.

Scientists today are paid to do science. Most of the profit generated from science comes from selling scientists equipment and kits. I have sat in meetings where a scientist will describe how he developed an assay. Step one, purchase assay. Step two, change a few buffers. Step three, use charts and graphs to describe the postive effects of the changes. Controls optional.

In Ben Franklins time it appeared that the scientists were the ones in the lab observing and doing experiements. They did not have to keep notebooks if they didn't want to. They occasionally caused themselves harm such as when Ben shocked himself unconcious accidentally while revving up enough volts to try and kill a turkey. Some scientists were extremely diligent when it came to measurements. Some were more qualitative when studying completely unknown phenomena. They all worked because so much was unknown and knowledge was ripe for the picking. Who wouldn't want to wake up, dream up ways of testing ideas and watch the results happen first hand? For this Ben Franklin gave up running his business and spent ten years pursuing. The end results were very successful and rewarding.

Back to biotechnology, the cargo cult science industry. You've got 100 billion dollars and PhDs from the finest universities. You've also got the research from the universities flowing out with zero resistance from the schools. There is money and there are the nations best educated people working every day to contribute to scientific progress. Where are the results?

In this Benjamin Franklin documentary there were dramatizations of Ben and his comrades standing around the lab watching experiments from start to finish. There was a parlor game that they performed where everyone would hold hands in a circle. At one end of the circle someone held the cathode. At the other, someone would touch the anode and everyone in the circle would get a cute little shock all at the same time. Watching everyone jump at the same time, some hair sticking up, was of great entertainment value in those days. People wanted to see the results first hand.

These days we have computers. We have computer software that sets up graphs, runs statistical analysis and so on. Meetings are always held in board rooms. Never, even in small companies, do PhD scientists hold meetings in laboratories where the work is done. No one goes to the laboratory bench to watch any part of an experiment. In the board room, computer screen up on the wall thanks to the latest technology from Microsoft, graphs are shown, one after the other. They are meant to explain the binding powers of antibodies or the knock-down effect of siRNA. They show the "score" of an arthritic set of mice, with and without the next big anti-arthritus drug.

In my previous job there were several boxes of dead mice sitting in jars of formaldehyde, waiting to be "scored" for their degrees of arthritus. The problem was that you didn't need to be a great scientist to tell that nothing had happened. The work would have taking weeks and very little would have came of it. Worst of all you would have the charts and graphs to prove it, blasted up on that board room wall where the scientists would look on with disapproval of these uninteresting results. They didn't want to watch the mice get the injections. They didn't want to monitor the disease progression. They didn't want to help score the mice. They wanted the results, up on the board with bar graphs. Arthritus was measured by the height of a bar on a graph sitting next to another bar that represents the drug affect. A smaller bar meant less arthritus.

They never did get the siRNA cure for arthritus. As in most science project they slowly let it fade away, hoping that no one noticed. As a corporation they do not have to tell the scientific community anything about their work. Failure is described to non-scientific investors and par for the course. Got to keep the pipeline diversitified, just like a stock portfolia. Some things just don't work. You understand. And slowly billions and billions of dollars disappear. It's not an easy living, but it beats getting in that lab and testing your ideas in front of others. That type of work requires real scientists who want to see what is happening. Like Ben.

Thursday, June 22, 2006

A story in the paper yesterday asked the question, is a college education still a good investment? On one hand, a college education is an experience that young people can have. You live on your own, with thousands of other kids. You go to classes and learn about things that you will be asked about on a test. On the other hand it's a very expensive four years of your life. You will have little time to earn money. At the same time the spending of money will be accelerated. You are hoping that later in life your earning potential will be accelerated. If your parents can help you are ahead of the game. If you are putting yourself through college this is a major risk of an investment. What guarentees are there that you will have a good job some day?

People like to bring up the fact that Bill Gates never finished college and now he's the riches guy in the world. The link to education and money however is pretty solid. One of the main reasons is that all higher paying jobs require a degree just to get your foot in the door. If you're like some people you have contacts through family members are other associations who can give you that little extra something that gets you the job. If not you are forced to pay your dues. Get the education and then get the rest of the package that will land you your high paying job. Bill Gates had the rest of the package to begin with.

What is the rest of the package? I have a degree in Biochemistry. During the course of my education I used a Mass Spectrometry machine to analyze the compounds that I produced in organic chemistry lab. When a potential employer wants someone with these skills however, I cannot use my education. They are looking for hands on experience. What they are saying is that my education was superficial. They want something real because you won't be taking tests on Mass Spec on the job. You will be using the machine for what is was intended. Education is thus a mere introduction to the world you might be working in. As time marches on during this rapid advancement in technology, is education keeping pace with it's Bachelors, Masters, and Doctorol programs?

Vocational schools teach people how to perform such jobs as mechanic, plumber and so on. Plumbing has remained the same for a long time. Cars have become a little more advanced. In most new cars you have to take your car back to the dealer where specialized mechanics can run computer diagnostics. They have specialized tools fit to particular models. I'm no expert but I am guessing that individuals with basic mechanic degrees from vocational schools have additional training required if they are to work in a dealers garage.

Feynman talked about educators assuming that what they were teaching was actually learned. In math you can teach a student that 2 plus 2 equals 4. In the real world, does the student understand that 2 apples and 2 oranges equal 4 pieces of fruit and not 4 apples? I see hiring managers these days requiring education and experience.

Demonstrated working knowledge of scientific principles and standard laboratory practices.

Familiarity with Virology and/or Immunology.

Knowledge and experience in the correct handling of hazardous and radioactive substances.

Does familiarity with virology and/or immunology mean that these two fields are almost the same? Does the scientific degree mean that you can have a degree in physics? There is more to this job description:

That's a lot of stuff to know in two years. All of these skills are taught in 4 year degrees but not in a four year physics degree. There are no degrees that train hiring managers to write up job descriptions. Human resource people are generally considered to be the experts here but this is not a science. It is an attempt to hire the right person for a a job. You could just say you are looking for someone with a degree in Biochemistry who can do some molecular biology work. RNAi? The field is too new to have any meaning. FACs? Just a machine that is complicated but not beyond the learning curve for a college grad. Development of assays? It's more of an art that people know or do not. I don't think it can be taught. Kits can be sold but developing an assay is real science. If we had an assay for cancer for example we would understand cancer much better.

The real issue of education and employment is the same as a Cargo Cult ceremony and airplanes landing. If you are in charge of hiring, do you know the difference between a typist and a secretary? If you are seeking employment do you know that having the skills and education is what you need to get an interview? Doing well at the interview gets you the job. The additional skills are what make the airplanes land. Many human endeavors fail because someone started with a lack of knowledge or finished with a lack of knowledge. True knowledge is what education should lead to. A college degree is a weak substitution for evidence of true knowledge.

Monday, June 12, 2006

When you work in a laboratory in the biomedical world you have to learn a few assays. An assay is a qualitative or quantitative analysis of a substance to determine its components. You put on your white lab coat, grab your pipettes and go do an assay. Are you doing science?

The cargo cult theory says that people will follow the form what they see others do in hopes of acheiving the same results. Scientists run assays so if you want to be a successful scientist someone had better get in that lab and run some assays. Let's say you are an office scientist and you do research on HIV. You will have to hire someone to work in the lab who knows how to test for the HIV virus. There will be an HIV assay. The fastest assay involves testing for antibodies against HIV. This is often the case for assays. You don't test for the component directly but for things associated with the component. The further you get away from the lab however, the less you know about the details of the assay.

The office scientist usually knows about the assay. An ELISA assay involves using an antibody against a protein. The antibody has an enzyme physically linked to it. The enzyme is tested for by adding a substrate that will create a color when cleaved by the enzyme. Remember you are really interested in the protein that is bound by the antibody that is linked to the enzyme that causes the substrate to make the color. How do you assess the significance of the simple development of a color?

The ELISA assay was developed long ago. Someone else did the work to determine that this assay could be used as an assay. Those who come along and choose to use it must determine of the things they are testing for can be used in a similar manner. Here is the cargo cult moment. An office scientist could instruct the young lab worker to run an ELISA assay to determine of protein X is present in some sample. The young lab worker buys a kit, runs the assay and looks for the presence of a color. This is similar to setting up an airport and looking to the skies for airplanes.

You have a sample that you suspect contains your protein. You want to know if it is there and how much of it is there. If you accidentally use too much antibody you will get a strong color. If you use too little you will get a weak color where a strong color should have been observed. You must have controls. If you want to know how much you must have a standard curve. The standard curve must involve y = mx + b and linear regression if it is to be understood quantitatively. Your standard curve must be prepared in the same medium as your sample. Your color must be read at the proper wavelenght. All samples must be stopped at the same time with the acid. If you were to seriously teach about the ELISA there would be more time spent on these issues than on any discussion involving antibodies and substrates. Light absorbance readings for example, have limitations. There is a linear range for reading the absorbance of a color in the machines used in the lab. That means there are limitations that must be known and accounted for. The standard curve is also a test of the limitations.

I bring all of this up because of the limitations of measurements in any science. The machine that measures the abosorbance of a colored solution has limitations. The ELISA assay has an upper and lower limit of detection no matter what you are measuring. That means there is a range where useful and accurate information can be obtained. Do you know what your range is?

Beyond ELISAs there are many assays used in basic biomedical research. They all have limitations that are woven together in a way that can cause great confusion. Even if you have 100% pure samples you will have a margin of error in your assay measurements. Imagine then trying to measure the amount of a protein in a blood sample or in kidney cells. Can you find an internal standard in blood? Are proteins sometimes upregulated and downregulated in times and concentrations that are out of our range to measure? I say hell yes they are yet we constantly read about people claiming to have accurately made the measurements nonetheless. Their measurements conveniently support their assumptions underlying the purpose of the research.

I once worked with RAW cells which are precursors to Osteoclasts. If you add RANK ligand to a RAW cell culture they will fuse together and form large multinucleated osteoclasts in about five days. I observed this process many times. I tested for the presence of tartrate resistant alkaline phosphatase (TRAP) every day. I could see TRAP on the surface of the Raw cells just after adding the RANK ligand. As the cells began to fuse I could see the TRAP on one side of the new ball of cells. By the fourth day the TRAP was nearly gone and the osteoclasts were predominant. These were simple observations. If I wanted to know more about TRAP and it's role in osteoclastogenisis I would have to get more clever than cell staining and microscopic observation. That's when I leave the cargo cult world and figure out what to do. I still have the lab coat and the microscope but I have to do something scientific. I have to know my limitations. I have to think about the location of the TRAP and how the cells pH or polarity might be affecting that location. How might the cells pH or polarity affect my measurements? In the end I would not propose any reasons for the TRAP regulation. I would merely describe what I think is going on. That's all. Next I would follow a lead that came up during the TRAP research. The goal would not be to cure osteoporosis. It's simply to observe nature.

It's like a reality show that only a few can watch. Do you get that channel? If so do you turn it on? If so is the picture fuzzy or clear? If it's clear do you have the ability to tell others about it? If so are you telling the right peope about it?

Friday, June 09, 2006

The Cargo Cult Airport has leaders. They send the people out with coconuts over their ears. They make sure the fires are lit along the side of the runway. If it's anything like the American corporate style of leadership, it is assumed that the leaders know more about why these things are done. The followers just follow. Leaders instruct because they are "in the know".

siRNA has been in existance a relatively short period of time. As soon as it began however, scientist began positioning themselves into the leadership role. There were rules set up for how to design siRNA. There were papers written on the best way to get the most out of your siRNA knock-out. Like a Cargo Cult Airport however, it wasn't known how siRNA really worked. The experts seemed have a disdain for research that involved a mechanism of action. One of "the rules" for example was that the siRNA had to consist of a certain percentage gaunisine and cytosine. What was the data used to come to that conclusion? Five siRNA KOs? Ten? Were they in mice or hamsters?

Eventually the scientists have to come up with a MOA, mechanism of action. It has been decided that the mechanism of action involves mRNA interacting with an enzyme called Dicer. One of the leading siRNA researchers, Dr. Kazunari Taira of the University of Tokyo describes it this way:

The mechanism responsible for dsRNA-induced gene silencing, which proceeds via a two-step mechanism, appears to have been strongly conserved during evolution. In the first step, long dsRNAs are recognized by a nuclease in the RNase III family known as Dicer, which cleaves the dsRNA into small interfering RNAs (siRNAs) of 21–23 nt. These siRNAs are incorporated into a multicomponent nuclease complex, known as RISC, that is then responsible for the destruction of cognate mRNAs.

Dr. Taira was a leader in siRNA research. More specifically he was ranked number 4 (most cited authors) out of the 25,000 researchers who have published papers on siRNA. He took on the toughest piece of the puzzle, the MOA and he got burned. A panel at the university where he worked said that he "likely fabricated papers on a potential medical breakthrough". As I've stated before, big time scientists do not work in laboratories. They work in offices. They communicate on the things that take place in labs but they rarely go inside their labs. One of the people who did work in the lab was Hiroaki Kawasaki. Dr. Kawasaki was most likely fabricating the data and Dr. Taira either couldn't figure that out or he was complicit in this scam.

The question now is how much this impacts the origins of the science. Does this leave a gap open where Dicers role is not known as a fact. Dr. Taira was saying that he could use recombinant Dicer to make siRNA that will work so that you don't have to experiment with different pieces to find the best one. This is one of his pre-conceived conclusions anyway. However, has anyone ever cloned dicer and shown it to chop up dsRNA? What type of dsRNA modifications are needed for Dicer to grab ahold of it and chop it up. Certainly Dicer isn't running amok inside a cell grabbing any dsRNA it finds and chopping it up. If it did we'd have a hard time making proteins. What regulates the balance between dsRNA breakdown and mRNA protein production? There are many enzymes that are very well understood. We use them for chopping up DNA for cloning genes. We know exactly where they cut the DNA. Where does Dicer cut the DNA? Is it based on size? If it is, then how does the siRNA shut down the genes so specifically. We know that synthisized RNA is a hit or miss gamble. Somehow one enzyme gets it right in many many different genes. How can this be.

This is science. How does siRNA work. What are the unanswered questions? What tiny little piece of the puzzle can we study? Dr. Taira is rightfully getting his work put under a spotlight. What about putting a spotlight on the research area he was working on. Why did they have to lie? Certainly it's important for these guys to publish but why didn't the area they were working on give them the kind of results they wanted? This is the Feynman Cargo Cult edict of bending over backwards to prove oneself wrong. They inadvertantly proved that they could not do what they thought they could do. They were ranked #4 in the siRNA business. In the end their theory did not provide them with the knowledge it would take to simply clone an enzyme and characterize it. Something is wrong here.

Sunday, June 04, 2006

Charles Bukowski worked for the Post Office. He would do his job and come home and drink wine and write poetry. He didn't care much for his day job but he loved staying up late drinking alone, listening to classical music on the radio and typing out poems. His canvas was a white sheet of paper shoved into an old type writer. The demons running around inside his head provided excellent material for his work. He was mad and said so. He wasn't trying to hide it. He was trying to tell someone about it in case someone wanted to help.

Those of us who love Bukowskis work refer to the man as Hank. Hank was a grizzly bear of a man. He loved to drink. He loved women. He hated working. Down at the post office you had to do what you're told. You had to take tests putting letters into slots based on the addresses on the envelopes. There were bad bosses and co-workers to deal with. One day Hanks publisher told him to quit the post office job. They worked out the amount of money needed to support Hanks lifestyle and it was provided by the publisher. It amounted to something like 105 dollars a month for rent, food, booze and cigarettes. At the age of 50 he quit his job and became a professional writer. Within the month he had written his first novel. He wrote several more novels and hundreds of poems. The novels served as background material that helped the reader understand where some of those poems were coming from. At the end of his life there was a body of work that came together and told a story.

I'd like to fashion this blog after people like Bukowski. I'd like to bring up the beatniks. In college they teach science and they teach art. So far it's been the arts that have allowed outsiders to make an impact on society. Bukowski was a postal worker. Kerouac was a vagabond. One thing the best of them all shared was a lack of formal education. They wanted to write so they did. A scientist however will have a hard time in the modern realm of the business. Many physical scientists for example, have a hard time renting the time on an atom smasher to prove their theories. It costs money. Writing a book does not. So what I'm trying to do with this blog is create a place where beatnik thinkers can contemplate the possibilities of the natural world. That's not science of the latest fashion. It's science that applied to people who existed in medieval times. Lacking the ability to smash atoms or look at cells left people with no other options than to dream up possibilities. Imagination was king. As I've mentioned before, marketing is now king in science.

Step away from the kings of science and sketch out a possibility that you've been thinking about for awhile. Go to the local pub and sit at a table by yourself and write a long paper that you won't have to worry about getting published. Invent a start-up company in your head and don't worry about the investors. The business of science is run by people who are the best at getting promoted. The best scientists are probably sitting at lab benches all day wondering about the data they get and how it really happens. They don't believe much of the hype coming out of the labs they work in but they are keenly interested in the experiements. The big time scientists have a story they need to tell and they are only excepting data that helps tell that story. The small time scientists are like poets. They wonder about the world they live in. They just don't know how to get anywhere with their ideas so they hold them in. To the poets of science I say, let it out. Write it down. Start a blog. Start a journal. Seek out other disgruntled workers and use your anger to strike at the status quo. Do something to them that they never did for you. Make them think. Make sure that at the end of your life there is a body of work that people can use.

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My biotech career is merely a dream. In this dream we are all living on an Island where our ancestors once watched the westerners develop drugs that helped their people fight off disease and suffering. The westerners left and we are now donning their white lab coats and trying to create the drugs ourselves. We have their books, labs, beakers and a handful of drug targets they left behind. But nothing is working.